Regulation of interleukin-2 signaling by fatty acids in human lymphocytes

  • Renata Gorjão
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
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  • Sandro Massao Hirabara
    Affiliations
    Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

    Program of Post-Graduation Studies in Physical Education, Center of Biological Sciences and Health, Cruzeiro do Sul University, São Paulo, Brazil
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  • Thaís Martins de Lima
    Affiliations
    Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
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  • Maria Fernanda Cury-Boaventura
    Affiliations
    Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

    Program of Post-Graduation Studies in Physical Education, Center of Biological Sciences and Health, Cruzeiro do Sul University, São Paulo, Brazil
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  • Rui Curi
    Affiliations
    Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
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      Docosahexaenoic (DHA; C22:6 n-3), eicosapentaenoic (EPA; C20:5 n-3), palmitic (PA; C16:0), and stearic (SA; C18:0) acids decrease lymphocyte proliferation in concentrations of >50 μM, as observed in our previous study. However, oleic acid (OA; C18:1 n-9) and linoleic acid (LA; C18:2 n-6) increase lymphocyte proliferation at 25 μM. In this study, the effect of these FAs on the interleukin-2 (IL-2) signaling pathway in human lymphocytes was investigated. Cells were isolated from heparinized venous blood of healthy human donors by density-gradient sedimentation. Cells were stimulated with 5 μg/ml concanavalin A and treated with FAs in the absence or presence of IL-2 for 1 hour. CD25-α externalization was analyzed by flow cytometry, and Janus kinase 1 (JAK1), JAK3, signal transducer and activator of transcription (STAT) 5, extracellular signal-regulated kinases (ERKs) 1 and 2, Akt, and protein kinase C (PKC)-ζ phosphorylation were analyzed by Western blotting. The expression of CD25-α at the cell surface was increased by DHA, SA, and PA but was unaffected by EPA, OA, and LA. PA, SA, DHA, and EPA decreased JAK1, JAK3, STAT5, and Akt phosphorylation induced by IL-2, but OA and LA did not cause any effect. OA and LA increased ERK1/2 phosphorylation, whereas the other FAs caused a marked decrease. PKC-ζ phosphorylation was decreased by OA and LA and was not altered by the remaining FAs. In conclusion, the inhibitory effect of PA, SA, DHA, and EPA on lymphocyte proliferation observed in our previous study was attributable to a decrease in JAK/STAT, ERK, and Akt pathways activated by IL-2. Probably, OA and LA stimulated lymphocyte proliferation by increasing ERK1/2 phosphorylation through PKC-ζ activation. The inhibition of JAK1, JAK3, STAT5, ERK1/2, and Akt phosphorylation caused by DHA, SA, and PA is associated with an alteration of CD25 expression at the cell surface.

      Abbreviations

      ConA
      concanavalin A
      DHA
      docosahexaenoic acid
      EPA
      eicosapentaenoic acid
      ERK
      extracellular signal-regulated kinase
      IL-2
      interleukin-2
      IL-2R
      interleukin-2 receptor
      JAK
      Janus kinase
      LA
      linoleic acid
      MAPK
      mitogen-activated protein kinase
      NF-κB
      nuclear factor κB
      OA
      oleic acid
      PA
      palmitic acid
      PI3K
      phosphatidylinositol 3-kinase
      PKC
      protein kinase C
      PMA
      phorbol myristate acetate
      SA
      stearic acid
      STAT
      signal transducer and activator of transcription
      Lymphocyte proliferation and growth is a tightly regulated process: the T-cell antigen receptor controls the G0/G1 cell cycle transition and stimulates T-cells to produce interleukin-2 (IL-2) and express interleukin-2 receptors (IL-2Rs). The IL-2-IL-2R interaction drives G1-S phase progression and T-cell clonal expansion (
      • Smith K.A.
      The interleukin 2 receptor.
      ). The high-affinity receptor IL-2R consists of three subunits, the α, β, and γ chains. The IL-2-induced heterodimerization of β and γ chains results in intermolecular transphosphorylation of their corresponding receptor associated Janus kinase 1 (JAK1) and JAK3 (
      • Kirken R.A.
      • Rui H.
      • Malabarba M.G.
      • Howard O.M.
      • Kawamura M.
      • O'Shea J.J.
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      Activation of JAK3, but not JAK1, is critical for IL-2-induced proliferation and STAT5 recruitment by a COOH-terminal region of the IL-2 receptor beta-chain.
      ,
      • Russell S.M.
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      • Noguchi M.
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      • Silvennoinen O.
      • et al.
      Interaction of IL-2R beta and gamma c chains with Jak1 and Jak3: implications for XSCID and XCID.
      ). Members of the STAT (signal transducer and activator of transcription) family of transcription factors, downstream effectors of the JAK, are also regulated by IL-2 (
      • Beadling C.
      • Guschin D.
      • Witthuhn B.A.
      • Ziemiecki A.
      • Ihle J.N.
      • Kerr I.M.
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      Activation of JAK kinases and STAT proteins by interleukin-2 and interferon alpha, but not the T cell antigen receptor, in human T lymphocytes.
      ). IL-2-induced tyrosine phosphorylation of STAT5 allows src homology 2 domain-mediated homodimerization or heterodimerization, with a resultant induction of nuclear migration and sequence-specific DNA binding by the STATs (
      • Darnell Jr., J.E.
      • Kerr I.M.
      • Stark G.R.
      Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.
      ). The STAT5 serine phosphorylation is rapidly induced by IL-2, and tyrosine phosphorylation is required for the transcriptional activity of this factor. The serine/threonine kinase inhibitor H7 clearly blocks IL-2-mediated signals by a mechanism not yet elucidated (
      • Beadling C.
      • Ng J.
      • Babbage J.W.
      • Cantrell D.A.
      Interleukin-2 activation of STAT5 requires the convergent action of tyrosine kinases and a serine/threonine kinase pathway distinct from the Raf1/ERK2 MAPK pathway.
      ). IL-2 signal transduction also involves the activation of other distinct signaling pathways: the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) cascade and the phosphatidylinositol 3-kinase (PI3K) signaling pathway. The activation of these two pathways is necessary for cell proliferation and differentiation.
      Fatty acids can control leukocyte functions such as cytokine production (
      • Costabile M.
      • Hii C.S.
      • Melino M.
      • Easton C.
      • Ferrante A.
      The immunomodulatory effects of novel beta-oxa, beta-thia, and gamma-thia polyunsaturated fatty acids on human T lymphocyte proliferation, cytokine production, and activation of protein kinase C and MAPKs.
      ), macrophage phagocytosis (
      • Kew S.
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      • Quinlan P.T.
      • Calder P.C.
      The effect of feeding structured triacylglycerols enriched in eicosapentaenoic or docosahexaenoic acids on murine splenocyte fatty acid composition and leucocyte phagocytosis.
      ), and lymphocyte proliferation (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ). Some FAs (e.g., n-3) are generally considered beneficial for human health because of their immunosuppressive effects in some autoimmune and inflammatory diseases (
      • Camuesco D.
      • Galvez J.
      • Nieto A.
      • Comalada M.
      • Rodriguez-Cabezas M.E.
      • Concha A.
      • Xaus J.
      • Zarzuelo A.
      Dietary olive oil supplemented with fish oil, rich in EPA and DHA (n-3) polyunsaturated fatty acids, attenuates colonic inflammation in rats with DSS-induced colitis.
      ,
      • Mills S.C.
      • Windsor A.C.
      • Knight S.C.
      The potential interactions between polyunsaturated fatty acids and colonic inflammatory processes.
      ,
      • Robinson D.R.
      • Xu L.L.
      • Tateno S.
      • Guo M.
      • Colvin R.B.
      Suppression of autoimmune disease by dietary n-3 fatty acids.
      ). The n-3 FAs eicosapentaenoic acid (EPA; 20:5 n-3) and docosahexaenoic acid (DHA; 22:6 n-3) cause a marked decrease in lymphocyte proliferation at 50 μM and promote an inhibition of cell cycle progression induced by IL-2 (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ). On the other hand, n-6 FAs have proinflammatory properties by increasing intercellular adhesion molecule-1 expression (
      • Young V.M.
      • Toborek M.
      • Yang F.
      • McClain C.J.
      • Hennig B.
      Effect of linoleic acid on endothelial cell inflammatory mediators.
      ), natural killer cell activity (
      • Pompeia C.
      • Lopes L.R.
      • Miyasaka C.K.
      • Procopio J.
      • Sannomiya P.
      • Curi R.
      Effect of fatty acids on leukocyte function.
      ), and IgE production (
      • Harbige L.S.
      Dietary n-6 and n-3 fatty acids in immunity and autoimmune disease.
      ). Linoleic acid (LA; C18:2 n-6) promotes an increase of lymphocyte proliferation at low concentration (25 μM) and increases the stimulatory effect of IL-2 on lymphocyte proliferation (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ).
      The effect of FAs on lymphocyte function has been studied extensively, but the mechanisms involved are still unclear. EPA and DHA have been shown to inhibit the MAPK pathway in Jurkat T-cells during an antigenic challenge. Denys and colleagues (
      • Denys A.
      • Hichami A.
      • Khan N.A.
      Eicosapentaenoic acid and docosahexaenoic acid modulate MAPK enzyme activity in human T-cells.
      ,
      • Denys A.
      • Hichami A.
      • Maume B.
      • Khan N.A.
      Docosahexaenoic acid modulates phorbol ester-induced activation of extracellular signal-regulated kinases 1 and 2 in NIH/3T3 cells.
      ,
      • Denys A.
      • Hichami A.
      • Khan N.A.
      Eicosapentaenoic acid and docosahexaenoic acid modulate MAPK (ERK1/ERK2) signaling in human T cells.
      ) showed that EPA and DHA decreased the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) induced by phorbol myristate acetate (PMA) and anti-CD3 antibodies in Jurkat T-cells. PMA is known to activate MAPKs via the protein kinase C (PKC) pathway (
      • Denys A.
      • Hichami A.
      • Khan N.A.
      n-3 PUFAs modulate T-cell activation via protein kinase C-alpha and -epsilon and the NF-kappaB signaling pathway.
      ). These results suggest that these FAs may exert their inhibitory action on ERK1/2 activation by reducing PKC activity. Denys, Hichami, and Khan (
      • Denys A.
      • Hichami A.
      • Khan N.A.
      n-3 PUFAs modulate T-cell activation via protein kinase C-alpha and -epsilon and the NF-kappaB signaling pathway.
      ) showed that DHA and EPA inhibit membrane recruitment of PKC-α and PKC-ε. These PKC isoforms are coupled to MAPK activation upstream of ERK1/2, which leads to a decrease in nuclear translocation of nuclear factor κB (NF-κB), resulting in an inhibition of IL-2 gene expression and cell proliferation. Other studies have demonstrated that these FAs may exert suppressive effects on the phosphorylation of other MAPKs. Zeyda et al. (
      • Zeyda M.
      • Szekeres A.B.
      • Saemann M.D.
      • Geyeregger R.
      • Stockinger H.
      • Zlabinger G.J.
      • Waldhausl W.
      • Stulnig T.M.
      Suppression of T cell signaling by polyunsaturated fatty acids: selectivity in inhibition of mitogen-activated protein kinase and nuclear factor activation.
      ) studied the effect of PUFAs on MAPK phosphorylation and the transcriptional activity of AP-1, NF-AT, and NF-κB in Jurkat and primary T-cells after 2 days of treatment. The authors found that PUFAs promote a highly selective inhibition of c-jun N-terminal kinase phosphorylation and activation, whereas phosphorylation of other MAPKs, such as p38 MAPK, remained essentially unaltered by PUFA treatment in Jurkat and peripheral blood T-cells. These studies suggest that PUFAs have a selective action on some intracellular proteins involved in T-cell activation.
      Studies involving the effects of FAs on signaling pathways activated by T-cell receptor stimulation have been performed (
      • Denys A.
      • Hichami A.
      • Maume B.
      • Khan N.A.
      Docosahexaenoic acid modulates phorbol ester-induced activation of extracellular signal-regulated kinases 1 and 2 in NIH/3T3 cells.
      ,
      • Denys A.
      • Hichami A.
      • Khan N.A.
      n-3 PUFAs modulate T-cell activation via protein kinase C-alpha and -epsilon and the NF-kappaB signaling pathway.
      ,
      • Zeyda M.
      • Szekeres A.B.
      • Saemann M.D.
      • Geyeregger R.
      • Stockinger H.
      • Zlabinger G.J.
      • Waldhausl W.
      • Stulnig T.M.
      Suppression of T cell signaling by polyunsaturated fatty acids: selectivity in inhibition of mitogen-activated protein kinase and nuclear factor activation.
      ,
      • Li Q.
      • Wang M.
      • Tan L.
      • Wang C.
      • Ma J.
      • Li N.
      • Li Y.
      • Xu G.
      • Li J.
      Docosahexaenoic acid changes lipid composition and interleukin-2 receptor signaling in membrane rafts.
      ). However, the effect of FAs on cytokine signaling pathways, such as IL-2 signaling, is still unclear. Proteins related to IL-2R-activated pathways may be modulated by FAs, which could explain some of their immunomodulatory effects. We have shown previously that DHA, EPA, palmitic acid (PA), and stearic acid (SA) decreased the stimulatory effect of IL-2 on lymphocyte proliferation, increasing the percentage of cells in G1 phase and decreasing the proportion of cells in S and G2/M phases after 48 h of treatment, whereas LA and oleic acid (OA) at low concentration (25 μM) caused an additive effect to IL-2, increasing cell proliferation (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ).
      In this study, we investigated whether the effects of FAs on human lymphocyte proliferation stimulated by IL-2 reported previously (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ) are related to alterations in the IL-2 signaling pathway. The effects of 25 μM OA (C18:1 n-9) and LA (C18:2 n-6) and 50 μM PA (C16:0), SA (C18:0), DHA (C22:6 n-3), and EPA (C20:5 n-3) on the IL-2 signaling pathway in human lymphocytes after 1 h of treatment were examined. These FA concentrations were chosen based on a previous study in which we observed that OA and LA at 25 μM increased lymphocyte proliferation. This finding is in opposition to that caused by the other FAs at 50 μM, leading to a decrease of cell proliferation that did not promote cell death (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ).

      MATERIALS AND METHODS

       Chemicals

      RPMI-1640 medium, HEPES, fetal bovine serum, penicillin, and streptomycin were obtained from Invitrogen (Carlsbad, CA). PA, SA, OA, LA, DHA, EPA, concanavalin A (ConA), Histopaque-1077, glutamine, and recombinant IL-2 were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-pJAK1 (Tyr 1022/1023), anti-pJAK3 (Tyr 980), anti-pSTAT5 (Tyr 694), anti-pSTAT5 (Ser 726), anti-pERK1/2 (Tyr 204), anti-pAkt (Ser 473), anti-JAK1, anti-JAK3, anti-STAT5, anti-ERK1/2, anti-Akt, anti-rabbit and anti-mouse antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-pPKC-ζ, anti-PKC-ζ, and anti-CD25 were obtained from Cell Signaling Technology (Beverly, MA). FITC-conjugated anti-CD25 antibody was purchased from Pharmingen-BD Biosciences (San Diego, CA). Salts for buffer preparation were from Labsynth (Diadema, Brazil).

       Study design

      The study was approved by the Ethical Committee of the Institute of Biomedical Sciences, University of São Paulo. The human blood for lymphocyte isolation was obtained from the Blood Bank of the Federal University of São Paulo. The blood was considered healthy after a routine laboratory analysis.

       Isolation of peripheral blood lymphocytes

      Peripheral blood lymphocytes were isolated as described previously (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ). Blood was diluted in PBS (1:1), and this suspension was layered on Histopaque-1077 and centrifuged for 30 min at 400 g at room temperature. Peripheral blood mononuclear cells (a mixture of monocytes and lymphocytes) were collected from the interphase, and erythrocytes were lysed with 150 mM NH4Cl, 10 mM NaHCO3, and 0.1 mM EDTA, pH 7.4, and washed once with PBS.
      The peripheral blood mononuclear cells were maintained in RPMI-1640 medium to allow the adherence of monocytes to the plates to obtain a pure lymphocyte suspension (∼98%).

       Culture conditions

      The cells were grown in culture flasks containing RPMI-1640 medium with 10% fetal bovine serum. This medium was supplemented with glutamine (2 mM), HEPES (20 mM), streptomycin (10,000 μg/ml), penicillin (10,000 IU/ml), and sodium bicarbonate (24 mM). Cells were grown in 25 ml flasks containing 1 × 106 cells/ml. The cells were kept in a humidified atmosphere at 37°C containing 5% CO2.

       Fatty acid treatment

      The assays to evaluate the effects of FAs on IL-2-activated intracellular signaling were performed by incubating the cells with 5 μg/ml ConA for 24 h to stimulate the expression of the α subunit of the IL-2R (CD25). Afterward, lymphocytes were washed with PBS and then cultured with different FAs in the presence and absence of IL-2 (30 ng/ml) for 1 h. This concentration of IL-2 was chosen because it promoted the highest response of lymphocyte proliferation (data not shown).
      The FAs were previously diluted in ethanol. The concentration of ethanol was always <0.5%. This concentration was not toxic to the cells (data not shown), as also reported by Siddiqui et al. (
      • Siddiqui R.A.
      • Jenski L.J.
      • Neff K.
      • Harvey K.
      • Kovacs R.J.
      • Stillwell W.
      Docosahexaenoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process.
      ). The concentrations of the FAs used were not toxic but caused significant effects on the proliferation of human lymphocytes: 50 μM for EPA, DHA, PA, and SA and 25 μM for OA and LA (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ).

       Western blotting

      Lymphocytes were incubated for 1 h as described above in the absence or presence of 30 ng/ml IL-2 and 50 μM DHA, EPA, SA, and PA or 25 μM OA and LA. After the incubation period, IL-2 stimulation was stopped by the addition of ice-cold PBS, and cells were pelleted by short centrifugation. Lymphocytes (1 × 107 cells) were homogenized in 120 μl of extraction buffer (100 mM Trizma, pH 7.5, 10 mM EDTA, 10% sodium dodecyl sulfate, 100 mM NaF, 10 mM sodium pyrophosphate, and 10 mM sodium orthovanadate) and immediately sonicated at 4°C for 60 s. Samples were boiled for 5 min and centrifuged at 13,000 g for 40 min at 4°C. Aliquots of the supernatants were used for the measurement of total protein content, as described by Bradford (
      • Bradford M.M.
      A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
      ). Equal amounts of proteins of each sample (40 μg) were diluted in Laemmli buffer containing dl-dithiothreitol (1 M), heated in a boiling-water bath for 3 min, and separated using a 12% SDS-polyacrylamide gel (
      • Shapiro A.L.
      • Vinuela E.
      • Maizel Jr, J.V.
      Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.
      ). Western blotting was carried out according to the method described by Towbin, Staehelin, and Gordon (
      • Towbin H.
      • Staehelin T.
      • Gordon J.
      Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
      ). The proteins on the gel were transferred to a nitrocellulose membrane at 120 V for 1 h. Nonspecific bonds were blocked by incubating the membranes with 5% defatted milk in basal solution (10 mM Trizma, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) at room temperature for 2 h. Membranes were washed three times for 10 min each in basal solution and then incubated in basal solution containing 3% defatted milk at room temperature for 3 h with the following antibodies: anti-pJAK1 (1:1,000 dilution, Tyr 1022/1023), anti-JAK1 (1:1,000), anti-pJAK3 (1:1,000, Tyr 980), anti-JAK3 (1:1,000), anti-pSTAT5 (1:500, Tyr 694) or anti-pSTAT5 (1:400, Ser 726), anti-STAT5 (1:1,000), anti-pERK1/2 (1:500, Tyr 204), anti-pAkt (1:500, Ser 473), anti-pPKC-ζ (1:1,000), and anti-PKC-ζ (1:1,000) or anti-CD25 (1:1,000). Membranes were washed again (three times for 10 min each) and incubated with the corresponding secondary antibody (1:10,000 dilution) linked to horseradish peroxidase in basal solution containing 1% defatted milk at room temperature for 1 h. After the washings, membranes were incubated with substrate for peroxidase and chemiluminescence enhancer (ECL Western Blotting System Kit) for 1 min and immediately exposed to X-ray film. Films were then developed in the conventional manner (
      • Towbin H.
      • Staehelin T.
      • Gordon J.
      Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
      ,
      • Hirabara S.M.
      • de Oliveira Carvalho C.R.
      • Mendonca J.R.
      • Haber E.Piltcher
      • Fernandes L.C.
      • Curi R.
      Palmitate acutely raises glycogen synthesis in rat soleus muscle by a mechanism that requires its metabolization (Randle cycle).
      ). Quantitative analysis of blots was performed using Scion Image software (Scion Corp., Frederick, MD).

       Flow cytometric analysis

      After 1 h of treatment with FA and IL-2, lymphocytes (1 × 106 cells) were resuspended in PBS and labeled with FITC-conjugated anti-CD25 antibody (1:50) (Pharmingen-BD Biosciences), and the cell suspension was incubated for 1 h at 4°C in the dark. Negative control cells were incubated with isotype-matched nonreactive IgG1 antibody. Afterward, these cells were washed with PBS and analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Juan, CA). Fluorescence was measured using FL1-H channel (green fluorescence = 530/30 nm). Ten thousand events were analyzed per experiment. Cells with FITC fluorescence were evaluated using Cell Quest software (Becton Dickinson).

      RESULTS

       JAK1, JAK3, and STAT5 phosphorylation

      The effects of IL-2 on JAK1 and JAK3 phosphorylation in human lymphocytes were evaluated for up to 90 min of incubation. The highest phosphorylation of JAK1 and JAK3 promoted by IL-2 was observed at 60 min (Fig. 1). DHA, EPA, SA, and PA at 50 μM caused marked decreases (85, 24, 58, and 75%, respectively) of JAK1 phosphorylation (Figs. 2, 3) induced by IL-2. On the other hand, OA and LA at 25 μM did not cause any effects. The total expression of the three proteins was not altered by the fatty acids.
      Figure thumbnail gr1
      Fig. 1.Time course of Janus kinase 1 (JAK1) and JAK3 phosphorylation in human lymphocytes stimulated with interleukin-2 (IL-2). Lymphocytes were stimulated with concanavalin A (ConA) for 24 h and then washed and incubated with 30 ng/ml IL-2 for 10, 20, 30, 60, and 90 min.
      Figure thumbnail gr2
      Fig. 2.Effects of 50 μM docosahexaenoic (DHA), eicosapentaenoic (EPA), stearic (SA), and palmitic (PA) acids and 25 μM oleic (OA) and linoleic (LA) acids on JAK1 phosphorylation induced by IL-2. After densitometry analysis, data were normalized to the respective controls, which received a value of 100% in each experiment. The values are presented as means ± SEM of four experiments. * P < 0.05, for comparison between the treatments with fatty acids versus control (in the absence of FAs and IL-2); P < 0.05, for comparison between the treatments with fatty acids versus control treated with IL-2.
      Figure thumbnail gr3
      Fig. 3.Effects of 50 μM DHA, EPA, SA, and PA and 25 μM OA and LA on IL-2-induced JAK3 phosphorylation. After densitometry analysis, data were normalized to the respective controls, which received a value of 100% in each experiment. The values are presented as means ± SEM of four experiments. * P < 0.05, for comparison between the treatments with fatty acids versus control (in the absence of FAs and IL-2); P < 0.05, for comparison between the treatments with fatty acids versus control treated with IL-2.
      DHA, EPA, PA, and SA caused decreases (42, 26, 69, and 27%, respectively) of STAT5 tyrosine residue phosphorylation induced by IL-2 (Fig. 4). Phosphorylation of serine residue was also decreased by EPA, DHA, PA, and SA (45, 52, 24, and 20%, respectively) (Fig. 5). Both residues (tyrosine and serine) must be phosphorylated for the highest transcriptional activity of STAT5 induced by IL-2 (
      • Beadling C.
      • Ng J.
      • Babbage J.W.
      • Cantrell D.A.
      Interleukin-2 activation of STAT5 requires the convergent action of tyrosine kinases and a serine/threonine kinase pathway distinct from the Raf1/ERK2 MAPK pathway.
      ). OA and LA did not cause significant effects (data not shown).
      Figure thumbnail gr4
      Fig. 4.Effects of 50 μM DHA, EPA, SA, and PA and 25 μM OA and LA on signal transducer and activator of transcription (STAT) 5 tyrosine 694 phosphorylation induced by IL-2. After densitometry analysis, data were normalized to the respective controls, which received a value of 100% in each experiment. The values are presented as means ± SEM of four experiments. * P < 0.05, for comparison between the treatments with fatty acids versus control (in the absence of FAs and IL-2); P < 0.05, for comparison between the treatments with fatty acids versus control treated with IL-2.
      Figure thumbnail gr5
      Fig. 5.Effects of 50 μM DHA, EPA, SA, and PA and 25 μM OA and LA on STAT5 serine 726 phosphorylation induced by IL-2. After densitometry analysis, data were normalized to the respective controls, which received a value of 100% in each experiment. The values are presented as means ± SEM of four experiments. * P < 0.05, for comparison between the treatments with fatty acids versus control (in the absence of FAs and IL-2); P < 0.05, for comparison between the treatments with fatty acids versus control treated with IL-2.

       ERK1/2 phosphorylation

      The phosphorylation of ERK1/2 induced by IL-2 was decreased (by 83% and 27%) by DHA and EPA treatment, respectively (Fig. 6). DHA also caused a decrease of 62% in cells not stimulated with IL-2. PA and SA promoted decreases of 64% and 38% in the phosphorylation of these proteins (Fig. 6). The total content of ERK1/2 was not changed by these FAs. On the other hand, OA and LA caused increases of ∼25% in the ERK1/2 phosphorylation induced by IL-2 (Fig. 6). LA also increased (by 62%) ERK1/2 phosphorylation in unstimulated cells.
      Figure thumbnail gr6
      Fig. 6.Effects of 50 μM DHA, EPA, SA, and PA and 25 μM OA and LA on extracellular signal-regulated kinase (ERK) 1/2 phosphorylation induced by IL-2. After densitometry analysis, data were normalized to the respective controls, which received a value of 100% in each experiment. The values are presented as means ± SEM of three determinations from four experiments. * P < 0.05, for comparison between the treatments with fatty acids versus control (in the absence of FAs and IL-2); P < 0.05, for comparison between the treatments with fatty acids versus control treated with IL-2.

       Akt phosphorylation

      DHA and EPA exerted inhibitory effects on Akt phosphorylation induced by IL-2 (decreases of 86% and 31%, respectively) (Fig. 7). PA and SA also decreased Akt phosphorylation in cells stimulated (decreases of 78% and 72%, respectively) or not stimulated (decreases of 62% and 75%, respectively) with IL-2. OA and LA did not alter Akt phosphorylation after 1 h of treatment (Fig. 7). This observation suggests that these FAs probably exert their stimulatory effect through the Ras/ERK1/2 pathway.
      Figure thumbnail gr7
      Fig. 7.Effects of 50 μM DHA, EPA, SA, and PA and 25 μM OA and LA on Akt phosphorylation induced by IL-2. After densitometry analysis, data were normalized to the respective controls, which received a value of 100% in each experiment. The values are presented as means ± SEM of four experiments. * P < 0.05, for comparison between the treatments with fatty acids versus control (in the absence of FAs and IL-2); P < 0.05, for comparison between the treatments with fatty acids versus control treated with IL-2.

       PKC-ζ phosphorylation

      OA and LA promoted stimulatory effects on PKC-ζ phosphorylation in the absence (increase of 50%) and presence (increase of 100%) of IL-2. The total expression of this protein was not altered by these FAs. PA, SA, DHA, and EPA did not alter PKC-ζ phosphorylation (Fig. 8).
      Figure thumbnail gr8
      Fig. 8.Effects of 50 μM DHA, EPA, SA, and PA and 25 μM OA and LA on protein kinase C (PKC)-ζ phosphorylation induced by IL-2. After densitometry analysis, data were normalized to the respective controls, which received a value of 100% in each experiment. The values are presented as means ± SEM of three experiments. * P < 0.05, for comparison between the treatments with fatty acids versus control (in the absence of FAs and IL-2); P < 0.05, for comparison between the treatments with fatty acids versus control treated with IL-2.

       Expression of IL-2R α-chain

      IL-2R α-chain (CD25) assembles with β and γ subunits to form the high-affinity IL-2R (
      • Taniguchi T.
      • Minami Y.
      The IL-2/IL-2 receptor system: a current overview.
      ). Induction of CD25 expression is required for IL-2-induced signaling events. The histograms in Fig. 9 show the intensity of the sample fluorescence, which corresponds to FITC emission. The inhibition of CD25 expression at the cell surface decreases the fluorescence signal. Only 7.5% of cells were CD25-positive before being stimulated with ConA. ConA-induced surface expression of CD25 was not altered by treatment with IL-2. A significant percentage of cells expressing CD25 were reduced by DHA, PA, and SA treatment compared with the control (Fig. 9). The percentage of cells that were CD25-positive decreased from 36.5 ± 5.4% in cells treated with IL-2 to 12.6 ± 0.9%, 12.4 ± 1.6%, and 11.8 ± 1.1% (expressed as means ± SEM) in cells treated with DHA, SA, and PA, respectively. EPA, OA, and LA had no significant effects on CD25 expression after 1 h of treatment (Fig. 9). The total expression of CD25 analyzed by Western blotting was not altered by FA treatment (data not shown).
      Figure thumbnail gr9
      Fig. 9.Effects of 50 μM DHA, EPA, SA, and PA and 25 μM OA and LA on CD25-α lymphocyte surface expression. Lymphocytes were stimulated with ConA for 24 h, washed, and incubated with 30 ng/ml IL-2 for 1 h. Cells were pelleted and labeled with FITC-conjugated anti-CD25 antibody and analyzed by flow cytometry. Negative control cells were incubated with a marked nonreactive control antibody. Histograms from 10,000 events are shown. Fluorescence was measured in channel FL1-H (green fluorescence = 530/30 nm). A: Representative histograms obtained by flow cytometry. B: Percentage of cells expressing CD25-α. The values are presented as means ± SEM of three determinations from four experiments. * P < 0.05, for comparison between the treatments with fatty acids versus control (in the absence of FAs and IL-2); P < 0.05, for comparison between the treatments with fatty acids versus control treated with IL-2.

      DISCUSSION

      It is widely known that treatment with tyrosine kinase inhibitors reduces lymphocyte proliferation (
      • Wang H.
      • Xie X.
      • Lu W.G.
      • Ye D.F.
      • Chen H.Z.
      • Li X.
      • Cheng Q.
      Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway.
      ). This finding indicates that JAK1 and JAK3 tyrosine phosphorylation induced by IL-2 plays an essential role in lymphocyte proliferation (
      • Wang H.
      • Xie X.
      • Lu W.G.
      • Ye D.F.
      • Chen H.Z.
      • Li X.
      • Cheng Q.
      Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway.
      ). The present study showed that some FAs inhibit the signaling pathway activated by this cytokine in human lymphocytes after a short period of treatment (1 h). A decrease of JAK1 and JAK3 phosphorylation was observed when human lymphocytes were treated with DHA, EPA, PA, and SA in the presence of IL-2. The reduction of JAK1 and JAK3 phosphorylation may be an important mechanism for the inhibition of lymphocyte proliferation induced by these FAs, as reported in our previous study (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ). The total content of these proteins was not altered by the FA treatment, indicating that these FAs inhibit lymphocyte proliferation by altering the protein phosphorylation state. IL-2 is particularly important for the development, expansion, and survival of T-cells, and inhibition of the signaling pathway of this cytokine results in severe immunodeficiency (
      • Gaffen S.L.
      • Lai S.Y.
      • Xu W.
      • Gouilleux F.
      • Groner B.
      • Goldsmith M.A.
      • Greene W.C.
      Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity.
      ). Thus, inhibition of both IL-2 production (
      • Terada S.
      • Takizawa M.
      • Yamamoto S.
      • Ezaki O.
      • Itakura H.
      • Akagawa K.S.
      Suppressive mechanisms of EPA on human T cell proliferation.
      ) and IL-2 action in lymphocytes markedly contributes to the potent suppressive effects of DHA and EPA on the function of these cells.
      Inhibition of IL-2-induced STAT5 phosphorylation by DHA, EPA, PA, and SA occurred at the level of tyrosine and serine residues (Figs. 3, 4). JAK3 activates JAK1, which is then able to phosphorylate the target tyrosine residues in STAT5 molecules docked with the β subunit of the IL-2R. Therefore, STAT5 activation occurs when JAK1 and JAK3 are phosphorylated. Once JAK phosphorylation was decreased by PA, SA, EPA, and DHA treatment, STAT5 phosphorylation was also inhibited (Fig. 4, 5). JAK-STAT signaling by IL-2 is critically important for immune and inflammatory responses and for T-cell expansion and differentiation. Inhibition of the signaling of this cytokine, as occurs in genetic deficiency of the γ subunit of JAK3, results in severe immunodeficiency. The STAT5 molecule does not appear to be the only protein involved in this process. In fact, deletion and mutational experiments of STAT5 DNA binding activity or the prevention of STAT5 activation did not block IL-2R mitogenic signaling (
      • Gaffen S.L.
      • Lai S.Y.
      • Xu W.
      • Gouilleux F.
      • Groner B.
      • Goldsmith M.A.
      • Greene W.C.
      Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity.
      ,
      • Fujii H.
      • Nakagawa Y.
      • Schindler U.
      • Kawahara A.
      • Mori H.
      • Gouilleux F.
      • Groner B.
      • Ihle J.N.
      • Minami Y.
      • Miyazaki T.
      • et al.
      Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission.
      ). Although STAT5 does not seem to be essential for mitogenesis, it is involved in the induction of genes required for cell proliferation and survival (
      • Lord J.D.
      • McIntosh B.C.
      • Greenberg P.D.
      • Nelson B.H.
      The IL-2 receptor promotes lymphocyte proliferation and induction of the c-myc, bcl-2, and bcl-x genes through the transactivation domain of Stat5.
      ,
      • Yu C.R.
      • Ortaldo J.R.
      • Curiel R.E.
      • Young H.A.
      • Anderson S.K.
      • Gosselin P.
      Role of a STAT binding site in the regulation of the human perforin promoter.
      ).
      JAK1 and JAK3 activation promotes the phosphorylation of tyrosine residues in the cytoplasmic tail of β and γ subunits of IL-2R. Ras is activated through the binding of the adaptor Shc to the tyrosine-phosphorylated receptor, which recruits the Grb2-Sos complex and activates the Raf-MEK-ERK pathway. EPA and DHA promoted a decrease of ERK1/2 phosphorylation stimulated by IL-2. This inhibitory effect is attributable to a reduction of JAK1 and JAK3 phosphorylation promoted by these FAs that possibly led to a decrease in the activity of ERK1/2 (or p42/p44 MAPK). Activated ERK1/2 phosphorylates various substrates in the cell compartments. ERK activation plays an essential role in cell growth and provides an integrated response: increases nucleotide synthesis, activates the transcription of many genes acting via transcription factors (e.g., Elk-1, Fos, AP-1, NF-AT, and c-myc) and chromatin phosphorylation, stimulates protein synthesis, and finally facilitates the formation of an active cyclin D-CDK4 complex, which is a rate-limiting step for cell growth (
      • Whitmarsh A.J.
      • Davis R.J.
      Regulation of transcription factor function by phosphorylation.
      ). Khan et al. (
      • Khan N.A.
      • Nishimura K.
      • Aires V.
      • Yamashita T.
      • Oaxaca-Castillo D.
      • Kashiwagi K.
      • Igarashi K.
      Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation.
      ) also showed that DHA arrested the progression from the late G1 to the S phase of the FM3A mouse mammary cancer cell cycle by decreasing ERK1/2 phosphorylation.
      Denys and colleagues (
      • Denys A.
      • Hichami A.
      • Khan N.A.
      Eicosapentaenoic acid and docosahexaenoic acid modulate MAPK enzyme activity in human T-cells.
      ,
      • Denys A.
      • Hichami A.
      • Maume B.
      • Khan N.A.
      Docosahexaenoic acid modulates phorbol ester-induced activation of extracellular signal-regulated kinases 1 and 2 in NIH/3T3 cells.
      ) showed that EPA and DHA inhibited ERK1/2 phosphorylation, but this occurred by decreasing PKC-β activation in PMA-stimulated Jurkat cells. In fact, PKC is the main pathway activated by PMA stimulation in these cells. In our study, these two FAs decreased JAK1 and JAK3 phosphorylation, a signaling pathway stimulated by IL-2 that also results in ERK1/2 activation. The saturated FA, PA, and SA decreased ERK phosphorylation stimulated by IL-2. Similarly, Hirabara et al. (2003) showed that PA decreased ERK1/2 phosphorylation induced by insulin in 1 h incubated rat soleus muscle.
      OA and LA enhanced the effect of IL-2 on ERK1/2 phosphorylation, suggesting that this pathway may be involved in the stimulation of lymphocyte proliferation promoted by these FAs, as demonstrated previously (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ). Cury-Boaventura et al. (
      • Cury-Boaventura M.F.
      • Gorjao R.
      • de Lima T.M.
      • Piva T.M.
      • Peres C.M.
      • Soriano F.G.
      • Curi R.
      Toxicity of a soybean oil emulsion on human lymphocytes and neutrophils.
      ) observed a decrease in the proliferation of lymphocytes from volunteers who received a lipid emulsion rich in LA. This effect was possibly attributable to the toxicity of the LA, whose plasma level was markedly increased. Thanasak et al. (
      • Thanasak J.
      • Muller K.E.
      • Dieleman S.J.
      • Hoek A.
      • Noordhuizen J.P.
      • Rutten V.P.
      Effects of polyunsaturated fatty acids on the proliferation of mitogen stimulated bovine peripheral blood mononuclear cells.
      ) also showed that the treatment of bovine lymphocytes with low concentrations of LA (<25 μM) increased lymphocyte proliferation, whereas higher concentrations of this fatty acid decreased it. The results presented here suggest that LA at low concentration acts as a proinflammatory agent by stimulating the MAPK cascade, leading to lymphocyte proliferation. Activation of PKC is a key signaling event for cell growth and proliferation in response to several mitogens. Cis-unsaturated fatty acids (e.g., OA) can activate PKC and, in turn, ERKs, as observed by others (
      • Lu G.
      • Morinelli T.A.
      • Meier K.E.
      • Rosenzweig S.A.
      • Egan B.M.
      Oleic acid-induced mitogenic signaling in vascular smooth muscle cells. A role for protein kinase C.
      ). OA more potently and completely activates the Ca2+-independent and atypical PKC isoforms (
      • Murakami K.
      • Chan S.Y.
      • Routtenberg A.
      Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids.
      ,
      • Khan W.A.
      • Blobe G.
      • Halpern A.
      • Taylor W.
      • Wetsel W.C.
      • Burns D.
      • Loomis C.
      • Hannun Y.A.
      Selective regulation of protein kinase C isoenzymes by oleic acid in human platelets.
      ) in platelets. PKC-ζ has been shown to activate ERK, being associated with mitogenesis in various cell lines (
      • Berra E.
      • Diaz-Meco M.T.
      • Dominguez I.
      • Municio M.M.
      • Sanz L.
      • Lozano J.
      • Chapkin R.S.
      • Moscat J.
      Protein kinase C zeta isoform is critical for mitogenic signal transduction.
      ). Other cis-fatty acids, such as LA, also fully activate PKC in the same manner (
      • Murakami K.
      • Chan S.Y.
      • Routtenberg A.
      Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids.
      ). In contrast, PKC-α, a Ca2+-dependent isoform, is less potently stimulated by OA (
      • Khan W.A.
      • Blobe G.
      • Halpern A.
      • Taylor W.
      • Wetsel W.C.
      • Burns D.
      • Loomis C.
      • Hannun Y.A.
      Selective regulation of protein kinase C isoenzymes by oleic acid in human platelets.
      ) and has been associated with the differentiation rather than the proliferation of vascular smooth muscle cells (
      • Haller H.
      • Lindschau C.
      • Quass P.
      • Distler A.
      • Luft F.C.
      Differentiation of vascular smooth muscle cells and the regulation of protein kinase C-alpha.
      ). In the present study, OA and LA increased PKC-ζ phosphorylation after 1 h of treatment. Once OA and LA did not alter JAK/STAT phosphorylation, activation of the novel and/or atypical PKC isoforms may explain the stimulatory effect of OA on ERK1/2 phosphorylation induced by IL-2.
      Akt phosphorylation was decreased by DHA, EPA, PA, and SA. The effect of DHA was more pronounced than that of the other FAs, whereas OA and LA did not affect this protein phosphorylation (Fig. 7). The activation of the PI3K pathway can potentiate proliferative signaling associated with STAT5 (
      • Moon J.J.
      • Nelson B.H.
      Phosphatidylinositol 3-kinase potentiates, but does not trigger, T cell proliferation mediated by the IL-2 receptor.
      ). PI3K may activate transcription factors that mediate events downstream of the G1 cell cycle checkpoint. Several Akt substrates are transcription factors or are involved directly in the regulation of gene transcription (
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      Cellular survival: a play in three Akts.
      ). Probably, this inhibitory effect on Akt phosphorylation and the JAK-STAT and ERK1/2 pathways induced by DHA, EPA, PA, and SA are responsible for the decreased lymphocyte proliferation observed in our previous study (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ).
      The α subunit of IL-2R (CD25-α) is undetectable in resting T-cells. The expression of this protein is triggered by antigens (
      • Robb R.J.
      • Smith K.A.
      Heterogeneity of human T-cell growth factor(s) due to variable glycosylation.
      ), a stimulus that can be mimicked by lectins such as ConA (
      • Kimura G.M.
      • Miller M.E.
      • Leake R.D.
      • Raghunathan R.
      • Cheung A.T.
      Reduced concanavalin A capping of neonatal polymorphonuclear leukocytes (PMNs).
      ). If a decrease in CD25-α externalization occurs, IL-2R-stimulated pathways are suppressed and lymphocyte proliferation is inhibited. DHA, PA, and SA decreased CD25-α content at the plasma membrane surface in lymphocytes after 1 h of treatment (Fig. 9), with no alteration in the total expression of CD25 (data not shown). The CD25 surface membrane expression is necessary to form the high-affinity IL-2R that actually activates the intracellular signal pathways (JAK-STAT, Ras-ERK1/2, and PI3K-Akt) leading to lymphocyte proliferation. Probably, the inhibition of JAK1 and JAK3, ERK1/2, and Akt phosphorylation promoted by DHA, PA, and SA is attributable to the decrease in CD25-α expression and content in the plasma membrane. Other studies showed that PUFAs can alter receptor localization on the cell surface after 24 h of treatment (
      • Li Q.
      • Wang M.
      • Tan L.
      • Wang C.
      • Ma J.
      • Li N.
      • Li Y.
      • Xu G.
      • Li J.
      Docosahexaenoic acid changes lipid composition and interleukin-2 receptor signaling in membrane rafts.
      ,
      • Schley P.D.
      • Brindley D.N.
      • Field C.J.
      (n-3) PUFA alter raft lipid composition and decrease epidermal growth factor receptor levels in lipid rafts of human breast cancer cells.
      ). However, a change in the content of this receptor subunit in the plasma membrane does not fully explain the effect of all FAs on IL-2 signaling. It is noteworthy that OA and LA stimulated ERK1/2 phosphorylation but did not change CD25 expression. Measurements using red-fluorescent Alexa Fluor 594 conjugate of cholera toxin subunit B showed that, except for DHA, none of the FAs tested caused marked changes in lipid raft distribution under the conditions of this study (data not shown). Therefore, the FAs tested hardly affected intracellular signaling through changes in lipid rafts.
      Although DHA and EPA have promoted an inhibitory effect on lymphocyte proliferation (
      • Gorjao R.
      • Cury-Boaventura M.F.
      • de Lima T.M.
      • Curi R.
      Regulation of human lymphocyte proliferation by fatty acids.
      ), DHA caused a more pronounced decrease of ERK1/2 and JAK1 phosphorylation in relation to EPA (Figs. 2, 6). Other studies have shown that different n-3 FAs can exert divergent effects on immune response. DHA-rich fish oil caused an increase in neutrophil and monocyte phagocytosis (
      • Gorjao R.
      • Verlengia R.
      • Lima T.M.
      • Soriano F.G.
      • Boaventura M.F.
      • Kanunfre C.C.
      • Peres C.M.
      • Sampaio S.C.
      • Otton R.
      • Folador A.
      • et al.
      Effect of docosahexaenoic acid-rich fish oil supplementation on human leukocyte function.
      ), whereas others demonstrated that EPA-rich fish oil did not alter this cell function (
      • Miles E.A.
      • Banerjee T.
      • Dooper M.M.
      • M'Rabet L.
      • Graus Y.M.
      • Calder P.C.
      The influence of different combinations of gamma-linolenic acid, stearidonic acid and EPA on immune function in healthy young male subjects.
      ). Verlengia et al. (
      • Verlengia R.
      • Gorjao R.
      • Kanunfre C.C.
      • Bordin S.
      • de Lima T.M.
      • Martins E.F.
      • Newsholme P.
      • Curi R.
      Effects of EPA and DHA on proliferation, cytokine production, and gene expression in Raji cells.
      ) showed that the effect of EPA on gene expression in a human B-lymphocyte cell line (Raji) was more pronounced (25.9% of genes investigated were altered) compared with that of DHA (8.4% of genes investigated were altered). We observed here that an alteration in lipid raft organization was induced by DHA, but it did not occur when lymphocytes were treated with EPA. The differences observed in the effects of these two fatty acids may occur because the spatial conformation of DHA is different from that of EPA as a result of its carbon length and degree of unsaturation. Therefore, we cannot generalize the effects of FAs from the same family on lymphocytes.
      In conclusion, the findings reported here are important for understanding the mechanisms by which different FAs alter lymphocyte proliferation. The inhibitory effect of PA, SA, and DHA on lymphocyte proliferation was associated with a reduction in the IL-2-induced activation of the JAK/STAT, ERK, and Akt pathways and a decrease in CD25-α expression. EPA decreased the phosphorylation of the IL-2R signaling proteins by a mechanism that did not involve CD25. Probably, OA and LA stimulated lymphocyte proliferation by increasing ERK1/2 phosphorylation through PKC-ζ activation.

      Acknowledgments

      The authors are indebted to J. R. Mendonça, G. de Souza, E. P. Portiolli, Dr. T. C. Alba-Loureiro, and A. S. Alves for technical assistance. The authors are grateful to Dr. José Augusto Barreto of the Blood Bank of the Federal University of São Paulo. This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, and Conselho Nacional de Desenvolvimento Científico e Tecnológico.

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          This article has been withdrawn by the authors: Regulation of interleukin-2 signaling by fatty acids in human lymphocytes. J. Lipid Res. 2007. 48: 2009–2019.
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